Method of production

ABSTRACT

The present invention in the field of oligosaccharide production provides a method of producing oligosaccharides of useful lengths without producing substantial amounts of monosaccharides and disaccharides (illustrated by FIG. 1). There is provided a method for producing an ingredient suitable for incorporation into a foodstuff, cosmetic, or nutraceutical, said ingredient comprising one or more oligosaccharides, wherein the oligosaccharides are produced in an enzymatic reaction, said enzymatic reaction comprising the step of contacting, in a solution or suspension, a polysaccharide-cleaving enzyme and a polysaccharide-containing feedstock, wherein said enzymatic reaction produces substantially no monosaccharides or disaccharides.

CROSS-REFERENCE

This application is a continuation application of InternationalApplication Serial No. PCT/EP2019/054380, filed Feb. 21, 2019, whichclaims priority to European Application Serial No. 18157957.4, filedFeb. 21, 2018, all of which each application is incorporated byreferenced in their entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Aug. 18, 2020, isnamed 56406_704_301_ST25.txt and is 41.6 kilobytes in size.

FIELD OF THE INVENTION

The invention relates to the enzymatic production of oligosaccharidesand their use in foodstuffs, cosmetics, and nutraceuticals.

BACKGROUND OF THE INVENTION

Sugary foods and drinks are an important part of culture and lifestylehabits across the world, but the sugar they contain has been linked toobesity, diabetes, poor dental health, and disruptive behaviour inpeople. Because of this, consumer preferences have been shifting awayfrom sugar-containing foods, and governments are increasinglyimplementing regulation to encourage the consumption of less sugar.

As such, industry has been searching for appropriate low-caloriesweeteners for many decades to substitute for sugar in food andbeverages. Unfortunately, many sugar substitutes are produced fromnon-natural resources, and often offer bitter undertones or otherunpleasant tastes along with their sweetness, both of which consumersfind unappealing. Moreover, while sweeteners are able to mimic thesweetness of sugar in food and drinks, few are able to mimic the otheraspects of sugar such as adding bulk, modulating texture, providingstructure, acting as a preservative, and modulating colour and flavourthrough caramelisation and Maillard reactions.

Dietary fibre is an important part of a positive diet, and helpsmaintain digestive health and a well-regulated gut flora. Such fibrecomprises polysaccharides of varying chain lengths and saccharide types.In addition to being found naturally in a wide spectrum of foods, fibrecan also be produced separately and added to other foods during theirmanufacture.

Methods of industrially producing dietary oligosaccharides may involvechemically or enzymatically cleaving long polysaccharides into shorterchains. However, in addition to chains of the desired length, mono- anddi-saccharides are liberated by this cleaving action. Because mono- anddi-saccharides are classed as ‘sugar’ in nutritional labelling, andbecause they cause the negative effects on human health described above,they are undesirable in many food uses for oligosaccharides. Glucose,galactose, fructose, maltose, sucrose and lactose in particular areundesired, as they are calorific. However, despite the negativeassociations with excess mono- and di-saccharides on human health,compositions comprising high levels of mono- and di-saccharides, such as100%, are abundantly used in the food industry.

SUMMARY OF THE INVENTION

The present inventor has found that sugar compositions comprising longerchained saccharides (oligosaccharides), which replace substantialamounts of the mono- and di-saccharides in the presently usedcompositions, still provide the desired sweetness and texture propertiesin a foodstuff. However, the negative effects that are associated withthe current sugar compositions on human health are significantlyimproved; for example, the compositions of the present invention containfar fewer calories and have less impact on dental health.

Furthermore, the present inventor has discovered enzymatic methods ofproducing oligosaccharides of useful lengths without producingsubstantial amounts of monosaccharides and disaccharides, and has foundthat foodstuffs derived from these oligosaccharides have improvedcharacteristics. Monosaccharides and disaccharides are often removedfrom oligosaccharide compositions, adding time, complexity, energy, andexpense to the manufacturing process. As a result, the inventor's novelmethods are useful in manufacturing foodstuffs, nutraceuticals, andcosmetic products.

Further, the inventor has found that when the enzyme is a LyticPolysaccharide Monooxygenase (LPMO), some of the oligosaccharide chainsproduced have chemical modifications at one or both termini which maymodulate the flavour, colour, caramelisation, and other properties ofthe oligosaccharide in such ways as are useful in the food industry.

According to a first aspect of the invention, there is provided a methodfor producing an ingredient suitable for incorporation into a foodstuff,cosmetic, or nutraceutical, said ingredient comprising one or moreoligosaccharides, wherein the oligosaccharides are produced in anenzymatic reaction, said enzymatic reaction comprising the step ofcontacting, in a solution or suspension, a polysaccharide-cleavingenzyme and a polysaccharide-containing feedstock, wherein said enzymaticreaction produces substantially no monosaccharides or disaccharides.

According to a second aspect of the invention, there is provided aningredient for incorporation into a foodstuff, cosmetic, ornutraceutical, comprising β-1,4-glucan oligosaccharides, wherein one ormore terminal saccharide residues are oxidised to a lactone, a4-ketoaldose, an aldonic acid or a geminal diol, and wherein theingredient comprises substantially no monosaccharides or disaccharides.

According to a third aspect of the invention, there is provided aningredient for incorporation into a foodstuff, cosmetic, ornutraceutical, comprising β-1,4-glucan oligosaccharides and anotheroligosaccharide.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: PACE gel showing products of incubation of phosphoricacid-swollen cellulose with a buffered solution of PaLPMO9E and/orascorbate, as per example 1.

FIG. 2: PACE gel showing products of incubation of washed oats with asolution of GH16 lichenase from Bacillus subtilis, as per example 2.

FIG. 3: Photo of cakes made by incorporating into the cake batter sugar,mixed-linkage glucan oligosaccharides or neither sugar noroligosaccharide, as per example 2.

FIG. 4: PACE gel showing products of incubation of spruce wood chipswith a buffered solution of GH30 xylanase from Ruminiclostridiumthermocellum, as per example 3.

FIG. 5: PACE gel showing products of incubation of tamarind xyloglucanwith a buffered solution of GH5 xyloglucanase from Paenibacillus sp, asper example 4.

DETAILED DESCRIPTION OF THE INVENTION

The inventor has discovered enzymatic methods of producingoligosaccharides of lengths useful in foodstuff, cosmetic, ornutraceutical products without also producing substantial amounts ofmonosaccharides and disaccharides. Some embodiments additionally offerproducts with novel properties.

As used herein, “food” and “foodstuff” refer to any item destined forconsumption, which may be consumption by a human, or by any otheranimal. It may be food, feed, a beverage, or an ingredient to be used inthe production of any of the above.

As used herein, “nutraceutical” refers to any composition introducedinto a human or other animal, whether by ingestion, injection,absorption, or any other method, for the purpose of providing nutritionto the human or other animal. Use in such a nutraceutical may take theform of a drink with added dietary fibre, a prebiotic additive, a pillor other capsule, tablet binding agent; or any other suitable use.

As used herein, “cosmetic” refers to any composition which is intendedfor use on humans or other animals to increase their aesthetic appeal orprevent future loss of aesthetic appeal, as well as any othercompositions known in general parlance as cosmetics. Aesthetic appeal isnot limited to visual aesthetics but applies as well to textural or anyother appeal. The cosmetic may be mascara, foundation, lip gloss,eyeshadow, eyeliner, primer, lipstick blush, nail polish, bronzer, orany other makeup; shampoo, conditioner, styling mousse, styling gel,hairspray, hair dye, hair wax, or any other hair product; moisturiser,exfoliant, suncream, cleanser, toothpaste, or a cream, a lotion,ointment or any other composition effective in modifying teeth, skin,hair or other parts of the body in some aesthetic way. Or it may be acomposition used as a component of a face mask, brush, hair roller,other styling device, or other solid structure, or any other suitablecomposition.

One step of the method of the current invention is an enzymaticreaction, in which one or more enzymes are placed in a suitable reactionvessel together with one or more feedstocks, which may be soluble orinsoluble in water, and a suitable solvent.

A variety of enzymes are suitable for use in the enzymatic reaction ofthe current invention. Any enzyme which, when acting on apolysaccharide-containing feedstock, produces oligosaccharides whileproducing substantially no monosaccharides or disaccharides may beappropriate. Preferably, the enzymatic reaction comprises a lyticpolysaccharide monooxygenase (LPMO), a lichenase, a xyloglucanendoglucanase (XEG), a mannanase, and/or a xylanase, such as a GH5, GH8,GH10, GH11 and/or GH30 xylanase. More preferably, the enzymatic reactioncomprises an LPMO. Even more preferably, the enzymatic reactioncomprises a mannanase. Yet more preferably, the enzymatic reactioncomprises a xylanase, such as GH5, GH8, GH10, GH11 or GH30 xylanase.Enzyme cocktails comprising numerous enzymes are also envisaged, forexample those comprising an LPMO and a xylanase, or those comprising anLPMO, a xylanase, and a lichenase or those comprising a xylanase and amannanase. Each enzyme may be provided to the enzymatic reaction as apurified enzyme, a semi-purified mixture derived from some naturalsource or lab-grown culture, in the form of a microbial strainengineered to produce the enzyme, or in any other way. Fusions of theseenzymes either with other enzymes or with non-enzymatic modules such ascarbohydrate-binding modules (CBMs) are also envisaged within eachrespective term, for example an LPMO fused to a CBM, a xylanase fused toa CBM, or a xylanase fused to an LPMO.

As used herein, “lytic polysaccharide monooxygenase” and “LPMO” refer toa class of enzymes able to oxidatively cleave polysaccharides using acopper-comprising moiety and using an oxygen source, such as a moleculeof dioxygen, peroxide, or any other oxygen source; and a suitablereducing agent. As such, when an LPMO is used, the enzymatic reactionmay be carried out under aerobic conditions. Suitable reducing agentsare not particularly limited, but examples include ascorbic acid, gallicacid, cysteine, NADH, NADPH, pyrogallol, dithiothreitol,cyanoborohydrides, borohydrides, photosynthetic pigments, lignin,lignols, and a combination of cellobiose and cellobiose dehydrogenase.While the skilled person knows a wide variety of photosynthetic pigmentswhich may be used, thylakoids and purified fractions, or chlorophyllin,are preferred, and light may be supplied.

The reducing agent is added to the enzymatic reaction at a certain molarconcentration ratio to the enzyme or enzyme cocktail. This ratio may beany suitable ratio, for example from about 10¹:1 to about 10⁸:1,preferably from about 10³:1 to about 10⁶:1, more preferably from about10⁴:1 to about 10⁵:1.

Aerobic conditions may comprise the addition of oxygen, which may beprovided by aeration of the substrate mixture with an oxygen-comprisinggas, such as air. Aeration may be conducted by the introduction ofoxygen-comprising air bubbles into the aqueous substrate mixtures byvarious systems, such as an air-injector, an aeration frit, a membranesystem, or an internal-loop airlift reactor. Preferably theconcentration of molecular oxygen in the enzymatic reaction is fromabout 4 to about 14 mg/I.

As the oxidising activity of LPMOs is particularly powerful, they canoxidatively cleave even very recalcitrant polymers such as cellulose.This makes production of useful oligosaccharides possible even fromfeedstocks which are seen traditionally as poor source materials forfood and are therefore very cheap. Examples of such feedstocks includeplant biomass such as grain, grain chaff, bean pods, seed-coats, and/orother seed materials; seaweeds; corn stover, corn cob, straw, bagasse,miscanthus, sorghum residue, switch grass, bamboo, and/or othermonocotyledonous tissue; water hyacinth, leaf tissue, roots, and/orother vegetative matter; hardwood, hardwood chips, hardwood pulp,softwood, softwood chips, softwood pulp, paper, paper pulp, cardboard,and/or other wood-based feedstocks; crab shells, squid biomass, shrimpshells, and/or other marine biomass; and/or any combination ofappropriate feedstocks. Feedstocks suitable for producing theoligosaccharide profile of the current invention when acted on by LPMOsmay comprise, for example, cellulose, chitin, chitosan, xylan and/ormannan, but any feedstock which can be suitably acted upon is envisaged.

Preferably, LPMOs are selected from the following families: AA9, AA10,AA11, AA13, AA14 and AA15. More preferably, the LPMO is PaLPMO9E (SEQ IDNO:1), an AA9 LPMO originally isolated from the ascomycete fungusPodospora anserina which produces particularly low levels ofmonosaccharides and disaccharides.

When LPMOs act on a substrate, of the two new terminal residuesgenerated in any given cleavage reaction, one is oxidised. When LPMOsare used, cellulose, chitin, and chitosan are preferred substrates. Ifcellulose, for example, is the substrate, when the β-1,4 glycosidic bondis cleaved, the residue attached to the C1 carbon is converted into alactone and the residue attached to the C4 carbon into a 4-ketoaldose.The two moieties may then spontaneously react with water to form analdonic acid and geminal diol respectively. The resultingoligosaccharides are thus largely equivalent to β-glucans generated inany other fashion, but differ subtly in some regards. Preferably theresulting oligosaccharides comprise β-glucans and/or polymers ofglucosamine.

In the case of glucans generated by LPMOs, the products may havedifferent caramelisation properties, flavour, colour, and otherproperties compared to equivalents generated via non-oxidising means. Assuch, while they can be used in the same applications as other glucans,they provide a subtle refinement in terms of these properties which maybe preferred to other sources of glucan in some applications. Similarly,use of different LPMOs yields different proportions of the differenttypes of oxidised ends and so use of different LPMOs can enable thetailoring of oxidation to suit different food, nutraceutical andcosmetic applications.

Another exemplary enzyme useful in the invention is a lichenase, whichmay be selected from the GHS, GH7, GH8, GH9, GH12, GH16, GH17, or GH26families, preferably a GH16 enzyme, more preferably a GH16 enzymederived from Bacillus subtilis (SEQ ID NO:2). Claimed herein is alichenase which produces substantially no monosaccharides ordisaccharides when acting on an appropriate polysaccharide substratesuch as lichenin or other mixed-linkage glucan. The enzyme is able toact on, for example, mixed linkage glucans, which are glucans comprisinga mixture of β-1,3 and β-1,4 linkages, and may cleave them at β-1,4glycosidic bonds. In the preferable case in which the lichenase acts ona mixed linkage glucan, the β-glucans produced may fall largely withinthe size range of from about 3 to about 7 residues, so they areparticularly useful in the food, cosmetics and nutraceutical industries.

Mixed linkage glucans are abundant in members of the grass and horsetailfamilies, and as such, grass-based feedstocks such as straw have highlevels of it, and may be acted upon usefully with lichenases.

Another alternative enzyme useful in the invention is a xylanase of theGHS, GH8, GH10, GH11 and/or GH30 family, which may act on, for example,feedstocks comprising a xylan backbone. The xylanase may be, forexample, a glucuronoxylanase, an arabinoxylanase, or aglucuronoarabinoxylanase. The enzyme may be active on a variety ofpolymers having a xylan backbone, such as glucuronoxylan, arabinoxylan,and glucuronoarabinoxylan. These polymers are abundant in variousplant-derived feedstocks, for example both hardwood and softwood maycomprise appropriate polysaccharides, with hardwood often comprisingglucuronoxylan and softwood often arabinoglucuronoxylan. Preferredxylanases include GH5 xylanases from Ruminiclostridium thermocellum (SEQID NO:3) and Gonapodya prolifera (SEQ ID NO:4), and GH30 xylanases fromDickeya chrysanthemi (SEQ ID NO:5), Bacillus subtilis (SEQ ID NO:6) andBacteroides ovatus (SEQ ID NO:7).

Feedstocks comprising softwood arabinoglucuronoxylan are preferredfeedstocks, and when digested with GH30 xylanases the products compriseoligosaccharides having a main chain of a length useful in thefoodstuff, cosmetics, and nutraceutical industries. Theseoligosaccharides may comprise more than about five main chain residuesand substantially no monosaccharides or disaccharides.

Feedstocks comprising hardwood glucuronoxylan are another preferredfeedstock, and when digested with GH30 xylanases the products compriseglucuronoxylan chains largely comprising from about 5 to about 30 mainchain residues.

Other enzymes useful in the invention include xyloglucanases andxyloglucan endoglucanases (XEGs), which are produced by numerousorganisms, including plant-pathogenic microbes. They are able to act onxyloglucan, a hemicellulosic β-1,4 glucan chain abundant in the primarycell wall of higher plants, which is decorated with xylose, some of thexylose residues being further decorated with other residues, such asgalactose. When appropriate xyloglucanases or XEGs act on xyloglucan,the products comprise xyloglucan oligosaccharides having a main chain ofa length useful in the foodstuff, cosmetics, and nutraceuticalindustries, and comprise substantially no monosaccharides ordisaccharides. One preferable xyloglucanase is a GH5 xyloglucanase fromBacteroides ovatus (SEQ ID NO:8).

The enzymatic reaction may take place in solution and/or suspension, ina suitable reaction vessel. At a temperature or temperature protocolappropriate for the particular combination of enzyme and feedstock, thereaction may be allowed to progress for a certain amount of time, oruntil the products have reached a desired concentration, or until someother requirement has been met.

As used herein, “suspension” refers to a composition comprising at leasttwo immiscible phases, for example, a solid and a liquid phase, whereinthe weight of the solid phase may be, as a percentage of the weight ofthe composition, in the range of from about 0.5% to about 30%,preferably 1% to about 10%, more preferably from about 2% to about 7%,yet more preferably from about 3% to about 5%. The suspension maycomprise a suitable solvent, which is preferably water. It may beparticularly beneficial to use a slightly higher concentration, forinstance to improve process time, of from about 1% to about 35%,preferably 5% to about 30%, more preferably from about 8% to about 25%,yet more preferably from about 10% to about 20%.

In order to ensure optimal contact between the enzymes and feedstock,the reaction mixture may be agitated, either constantly or at intervals.The agitation may take the form of rhythmically moving the entirereaction vessel, of a fan or other stirring device, of a bubblesparging, or any other method of agitation.

The enzymatic reaction may be a microbial fermentation. The temperatureand reaction time will be suitable for the growth of the microbialorganism used. The microbial organism may be genetically altered toproduce an enzyme suitable for the production of an oligosaccharide ofthe present invention, while producing substantially no monosaccharidesor disaccharides. The microbe may be, for example, a bacterium, forexample Escherichia coli, or a fungus, such as Saccharomyces cerevisiae.

Further embodied in the present invention is an expression vectorsuitable for modifying the subject microorganism such that it producesan enzyme or mixture of enzymes of the current invention. Where desired,the expression vector, which may be a plasmid or any other nucleic acidable to induce production of the enzyme, may comprise one or more of thefollowing regulatory sequences so as to control the expression of theexogenous enzyme: regulatory sequences of a heat shock gene, regulatorysequences of a toxicity gene, and regulatory sequences of a sporeformation gene.

The enzymatic reaction is carried out at a temperature or temperatureprotocol appropriate to the enzymes and substrates used. For example, itmay be carried out at a constant temperature in the range of from about10° C. to about 80° C., preferably about 20° C. to about 60° C., morepreferably from about 30° C. to about 40° C. It may be particularlybeneficial to use a slightly higher temperature, for instance to improveprocess time, of about 30° C. to about 70° C., preferably from about 40°C. to about 60° C. If the enzymatic reaction takes the form of amicrobial fermentation the temperature may be appropriate for such, forexample the enzymatic reaction may comprise the growth of E. coli and/orthe temperature may be constant and approximately 37° C.

The pH of the solution or suspension may affect the activity of theenzymes. Control of pH may be important in assuring that an enzymaticreaction proceeds at a suitable rate. The enzymatic reaction of thepresent invention may take place at a pH in the range of from about 2 toabout 10, preferably about 3 to about 8, more preferably about 4 toabout 6.

The enzymatic reaction is allowed to continue for a certain time periodbefore optionally being quenched, and the products isolated or otherwisecollected. This time period may be from about 1 minute to about 5 days,and is preferably from about 0.5 days to about 3 days, more preferablyfrom about 16 hours to about 48 hours. The reaction may alternatively beallowed to proceed until completion or approximate completion of thereaction. If the reaction is allowed to continue until completion orapproximate completion of the reaction, this may be longer than 5 days.

The one or more feedstocks added to the enzymatic reaction comprisepolysaccharides. Such polysaccharides may have been produced by aseparate reaction proceeding simultaneously in the reaction vessel. Thepolysaccharides present in the enzymatic reaction are cleaved by enzymesinto useful oligosaccharides.

Any substance which comprises appropriate polysaccharides may form partof the feedstock. As the foodstuff, cosmetic, and nutraceuticalindustries use a broad variety of oligosaccharides, the polysaccharidesappropriate for taking part in the enzymatic reaction are notparticularly limited. Preferably, the feedstock comprises one or morepolysaccharide selected from cellulose, chitin, chitosan, mixed-linkageglucan, xylan, and xyloglucan. If xylans are present, they preferablycomprise glucuronoxylan, arabinoxylan, and/or glucuronoarabinoxylan.

The feedstocks comprising such polysaccharides are also not particularlylimited, as most plant matter is rich in such polymers. As such, thefeedstock may comprise plant biomass such as grain, grain chaff, beanpods, seed-coats, and/or other seed materials; seaweeds; corn stover,corn cob, straw, bagasse, miscanthus, sorghum residue, switch grass,bamboo, and/or other monocotyledonous tissue; water hyacinth, leaftissue, roots, and/or other vegetative matter; hardwood, hardwood chips,hardwood pulp, softwood, softwood chips, softwood pulp, paper, paperpulp, cardboard, and/or other wood-based feedstocks; crab shells, squidbiomass, shrimp shells, and/or other marine biomass, and/or anycombination of appropriate feedstocks. Preferably, the feedstockcomprises wheat straw or wood. As any given natural feedstock is likelyto comprise a mixture of different polysaccharides, it will sometimes bethe case that a cocktail of different enzymes is beneficial. Such acocktail may comprise any other enzyme. For example, such a cocktailmight comprise a cellulase with a xylanase, a cellulase with amannanase, a xylanase with a mannanase, an LPMO with a xylanase, an LPMOwith a lichenase, an LPMO with a mannanase, or an LPMO with a differentLPMO in which the enzyme partners are present in molar ratios preferablybetween 1:10 and 10:1. In addition, as many appropriate feedstocks arerecalcitrant, pre-treatment of the feedstock is envisaged.

As used herein, “pre-treatment” is any process which makes a feedstockmore easily acted upon by the enzymes inherent in the enzymatic reactionstep of the current invention. The pre-treatment occurs before theenzymatic reaction, and may comprise acid treatment by, for example,sulphuric acid, phosphoric acid, or trifluoroacetic acid; alkalitreatment by, for example, sodium hydroxide, or ammonia fibre expansion;heat treatment by, for example, hot water, hot steam, or hot acid;and/or enzyme treatment by, for example, a hydrolase, lyase, or LPMO, orany mixture of the above processes.

As used herein, “polysaccharide” refers to a saccharide polymer of anylength greater than two residues. Polysaccharides may be highlybranched, lightly branched, or unbranched, may comprise any manner ofglycosidic bond in any combination, any number of, for example, α or βlinkages, and any combination of monomer types, such as glucose,glucosamine, mannose, xylose, galactose, fucose, fructose, glucuronicacid, arabinose, or derivatives thereof such as any combination of theabove monomers decorated with acetyl or other groups. The polysaccharidemay be a cellulosic or hemicellulosic polymer, hemicellulosic polymersenvisaged including xylan, glucuronoxylan, arabinoxylan, glucomannan,and xyloglucan. Cellulose is the preferred cellulosic polymer. Mannan ispreferred even more so. Xylan is preferred yet more still.

As used herein “highly branched”, “lightly branched”, and “unbranched”refer to the number of side-chains per stretch of main chain in asaccharide. Highly branched saccharides have on average from 4 to 10side chains per 10 main-chain residues, slightly branched saccharideshave on average from 1 to 3 side chains per 10 main-chain residues, andunbranched saccharides have only one main chain and no side chains. Theaverage is calculated by dividing the number of side chains in asaccharide by the number of main-chain residues.

As used herein, “saccharide” refers to any polysaccharide,oligosaccharide, monosaccharide, or disaccharide.

As used herein, “oligosaccharide” refers to saccharide polymers havingchain lengths generally within the range which is useful in the contextof a foodstuff, cosmetic, or nutraceutical product. They are comprisedat least within the products of the enzymatic reaction. Typical chainlengths may be from about 3 to about 16 saccharide residues.Oligosaccharides may be highly branched, lightly branched, orunbranched, may comprise glycosidic bonds in any combination, any numberof α or β linkages, and any combination of monomer types, such asglucose, glucosamine, mannose, xylose, galactose, fucose, fructose,glucuronic acid, arabinose, or derivatives thereof. Suitable derivativesinclude the above monomers comprising acetyl or other groups.

The oligosaccharides produced in the process of the present inventionfall within an upper and a lower size limit. The lower size limit isthat substantially no monosaccharides or disaccharides are produced.

As used herein, “substantially no” monosaccharides or disaccharidesrefers to a set of products in which by weight less than about 60%,preferably less than about 50%, preferably less than about 40%, morepreferably less than about 30%, even more preferably less than about20%, even more preferably less than about 15%, even more preferably lessthan about 10%, even more preferably less than about 5%, even morepreferably less than about 2%, yet more preferably less than about 1%,most preferably less than about 0.1%, of the imageable saccharides aremonosaccharides or disaccharides.

As described herein, the enzymatic reaction of the invention is usefulto produce oligosaccharides whilst producing substantially nomonosaccharides and disaccharides. However, it is envisaged that thereaction will take place in a large vessel with other reactions (e.g.enzymatic) taking place at the same time. These other enzymaticreactions will also be breaking down polysaccharides into smallersaccharides, including oligosaccharides, but may also producemonosaccharides and disaccharides. Thus, the method further comprises asecond enzymatic reaction comprising contacting a secondpolysaccharide-cleaving enzyme to the one or morepolysaccharide-containing feedstocks, which may produce one or moredisaccharides. In some instances monosaccharides may also be produced.These monosaccharides and disaccharides may be included in theingredient, thus in a specific feature, suitably the amount ofdisaccharides in the produced ingredient is less than about 50%,preferably less than about 40%, more preferably less than about 35%,more preferably less than about 30%, even more preferably less thanabout 25%, even more preferably less than about 20%, even morepreferably less than about 15%, even more preferably less than about10%, yet even more preferably less than about 5% of the imageablesaccharides.

Suitably the amount of monosaccharides in the produced ingredient isless than about 25%, preferably less than about 20%, more preferablyless than about 15%, even more preferably less than about 10%, even morepreferably less than about 5%, yet even more preferably less than about3%, yet even more preferably less than about 1% of the imageablesaccharides.

As used herein, “imageable polysaccharides” are those which are visiblein the gel or spectrum when one of the following imaging protocols iscarried out.

One way of assessing the percentages by weight of differentpolysaccharides produced by the current invention is processing a sampleof the enzymatic reaction products to derivatise their reducing endswith a fluorophore followed by polyacrylamide gel electrophoresis,before imaging the resulting polyacrylamide gel, for example byfluorescence imaging, and conducting optical density analysis on eachband, the resulting value to be adjusted by residue-count to give anindication of mass. The skilled person will be able to carry this outwith the information inside this application, in conjunction with Goubetet al. (2002). This is the method envisaged for calculating percentagevalues by weight of imageable polysaccharides.

Another way of assessing the percentages by weight of differentpolysaccharides produced by the current invention is to analyse byhigh-throughput liquid chromatography, for example using an anionexchange chromatography column in an alkaline solution, followed bypulsed amperometric detection. The resulting data can be adjusted byresidue-count to give an indication of mass. The skilled person will beable to carry this out with the information inside this application, inconjunction with Simmons et al. (2013).

As used herein “monosaccharide” and “disaccharide” refer to saccharidecompounds consisting respectively of one or two residues.Monosaccharides are compounds such as glucose, glucosamine, xylose,galactose, fucose, fructose, glucuronic acid, arabinose, galacturonicacid; or epimers or other derivatives thereof. Suitable derivativesinclude acetyl or other groups. Disaccharides are compounds consistingof two monosaccharides joined via any glycosidic bond. Envisaged hereinare enzymes or combinations of enzymes producing substantially nomonosaccharides or disaccharides in such a reaction.

The upper size limit of the oligosaccharides depends on the enzymes,feedstock, and reaction conditions used, and may be that the weight ofproducts comprising 16 or more residues in their main chain is below acertain percentage of the weight of imageable polysaccharides.

This percentage may be about 15%, preferably less than about 10%, morepreferably less than about 5%, even more preferably less than about 2%,most preferably less than about 1%; or, it may be that the weight ofproducts comprising 15 or more residues in their main chain is belowabout 15%, preferably less than about 10%, more preferably less thanabout 5%, even more preferably less than about 2%, most preferably lessthan about 1%, of the weight of imageable polysaccharides; or it may bethat the weight of products comprising 14 or more residues in their mainchain is below about 15%, preferably less than about 10%, morepreferably less than about 5%, even more preferably less than about 2%,most preferably less than about 1%, of the weight of imageablepolysaccharides, or, in increasing order of preference, that this is thecase with products comprising 13, 12, 11, 10, 9, 8, or 7 residues.

The feedstock may comprise cellulose, and when acted on by LPMOs orother enzymes, the weight of products comprising 7 or more residues intheir main chain may be below about 15%, preferably less than about 10%,more preferably less than about 5%, even more preferably less than about2%, most preferably less than about 1%, of the weight of imageablepolysaccharides. Or it may be that the weight of products comprising 8or more residues in their main chain may be below about 15%, preferablyless than about 10%, more preferably less than about 5%, even morepreferably less than about 2%, most preferably less than about 1%, ofthe weight of imageable polysaccharides.

The feedstock may comprise chitin, and when acted on by LPMOs or otherenzymes, the weight of products comprising 11 or more residues in theirmain chain may be below about 15%, preferably less than about 10%, morepreferably less than about 5%, even more preferably less than about 2%,most preferably less than about 1%, of the weight of imageablepolysaccharides. Or it may be that the weight of products comprising 12or more residues in their main chain may be below about 15%, preferablyless than about 10%, more preferably less than about 5%, even morepreferably less than about 2%, most preferably less than about 1%, ofthe weight of imageable polysaccharides.

The feedstock may comprise chitin, and when acted on by LPMOs or otherenzymes, the weight of products having only 3 or fewer residues in theirmain chain may be below about 15%, preferably less than about 10%, morepreferably less than about 5%, even more preferably less than about 2%,most preferably less than about 1%, of the weight of imageablepolysaccharides.

The feedstock may comprise mixed-linkage glucan, and when acted on bylichenase or other enzymes, the weight of products comprising 6 or moreresidues in their main chain may be below about 15%, preferably lessthan about 10%, more preferably less than about 5%, even more preferablyless than about 2%, most preferably less than about 1%, of the weight ofimageable polysaccharides. Or it may be that the weight of productscomprising 7 or more residues in their main chain may be below about15%, preferably less than about 10%, more preferably less than about 5%,even more preferably less than about 2%, most preferably less than about1%, of the weight of imageable polysaccharides.

The feedstock may comprise xylan, preferably glucuronoxylan,arabinoxylan, or arabinoglucuronoxylan, more preferably hardwoodglucuronoxylan or softwood arabinoglucuronoxylan.

The xylan may comprise arabinoglucuronoxylan, preferably softwoodarabinoglucuronoxylan, and when acted on by a xylanase, such as a GH30xylanase, or other enzyme, the weight of products comprising 9 or moreresidues in their main chain may be below about 15%, preferably lessthan about 10%, more preferably less than about 5%, even more preferablyless than about 2%, most preferably less than about 1%, of the weight ofimageable polysaccharides. Or it may be that the weight of productscomprising 10 or more residues in their main chain may be below about15%, preferably less than about 10%, more preferably less than about 5%,even more preferably less than about 2%, most preferably less than about1%, of the weight of imageable polysaccharides.

The xylan may comprise arabinoglucuronoxylan, preferably softwoodarabinoglucuronoxylan, and when acted on by a xylanase, such as a GH30xylanase, or other enzyme, the weight of products having only 5 or fewerresidues in their main chain may be below about 15%, preferably lessthan about 10%, more preferably less than about 5%, even more preferablyless than about 2%, most preferably less than about 1%, of the weight ofimageable polysaccharides.

The feedstock may comprise glucuronoxylan, preferably hardwoodglucuronoxylan, and when acted on by xylanase, such as a GH30 xylanase,or another enzyme, the weight of products comprising 31 or more residuesin their main chain may be below about 15%, preferably less than about10%, more preferably less than about 5%, even more preferably less thanabout 2%, most preferably less than about 1%, of the weight of imageablepolysaccharides.

The feedstock may comprise hardwood glucuronoxylan, preferably hardwoodglucuronoxylan, and when acted on by xylanase, such as a GH30 xylanase,or another enzyme, the weight of products having only 4 or fewerresidues in their main chain may be below about 15%, preferably lessthan about 10%, more preferably less than about 5%, even more preferablyless than about 2%, most preferably less than about 1%, of the weight ofimageable polysaccharides.

The feedstock may comprise xyloglucan, and when acted on by XEG or otherenzymes, the weight of products comprising 6 or more residues in theirmain chain may be below about 15%, preferably less than about 10%, morepreferably less than about 5%, even more preferably less than about 2%,most preferably less than about 1%, of the weight of imageablepolysaccharides. Or it may be that the weight of products comprising 7or more residues in their main chain may be below about 15%, preferablyless than about 10%, more preferably less than about 5%, even morepreferably less than about 2%, most preferably less than about 1%, ofthe weight of imageable polysaccharides

Where branched polymers are being described in terms of residue count,the number of residues refers only to the longest chain of residues, anddoes not include any side chains.

After the enzymatic reaction has progressed to a desired point, theproducts may be handled in a variety of ways. As the reaction mixturewill often comprise a mixture of soluble and insoluble products, with atleast some of the original feedstock often also remaining, the reactionmixture may be filtered to remove insoluble matter and prepare thesoluble products for further processing.

When used herein and otherwise unqualified, “soluble”, “solubility” andgrammatical variants refer to solubility in water.

The desired oligosaccharides may also be isolated from the enzymaticreaction mixture in a number of ways. They may be isolated based onsolubility, so that a composition of soluble saccharides only isextracted for further processing, and/or isolated chromatographically toproduce a composition with a narrower band of oligosaccharide chainlengths. Isolation may for example be based on precipitation,size-exclusion chromatography, ion-exchange chromatography, orfiltration, including ultrafiltration and nanofiltration. In the casethat isolation based on solubility is carried out, the profile ofsaccharides present in the isolated composition will depend on theoriginal enzymatic reaction, as different polysaccharides decrease insolubility with length at different rates.

Also envisaged in the scope of the invention is the further treatment ofthe produced oligosaccharides to produce further products beforeincorporation into a foodstuff, cosmetic, or nutraceutical. This furthertreatment may comprise any chemical, physical, or enzymatic step, suchas reduction, preferably reductive amination where appropriate;oxidation, caramelisation, modification with a Schiff base, or via theMaillard reaction, or by any combination of such steps, and may providedifferent products having properties which are improved for the desiredpurpose. For example the caramelisation properties, calorific value,flavour, and colour may be modified.

The products of the one or more enzymatic reactions may be deemed aningredient suitable for incorporation into a foodstuff, cosmetic, ornutraceutical at any stage of this process. For example, the reactionmixture itself, after the desired time limit or other condition forcompletion has been met, may directly be deemed the ingredient, oreither the solid or liquid component of the filtered products may be theingredient, or the composition of isolated oligosaccharides may be theingredient, or the oligosaccharides having undergone further treatmentmay be the ingredient.

As used herein, “ingredient” is any composition suitable forincorporation into a foodstuff, cosmetic, or nutraceutical product,which may include those which are used directly as the product itself.

The present ingredient suitable for incorporation into a foodstuff,cosmetic, or nutraceutical may be usable directly as a foodstuff,cosmetic, or nutraceutical product, or it may be mixed with otheringredients to form a foodstuff, cosmetic, or nutraceutical. Theingredient may also be treated in some physical or chemical way beforeor during incorporation into a foodstuff, cosmetic, or nutraceutical. Itmay be directly incorporated into a product, or it may be incorporatedinto, for example, a dough, cake mixture, chocolate mixture or otherfood precursor; a cosmetic base composition; or a nutraceutical, and beoptionally cooked or otherwise treated in a way which may cause chemicalmodification, a change of texture, a change of colour, or othermodification.

Once a composition of the oligosaccharide products suitable for theapplication being considered is obtained, and further treatment and/orisolation is optionally carried out, the derivation of a foodstuff,cosmetic, or nutraceutical from the composition furnishes a very broadarray of potential uses. The ingredients of the current invention areuseful in applications in which oligosaccharides are conventionallyused. They are particularly useful in applications in whichmonosaccharides and disaccharides are detrimental and would otherwise beconsidered for removal.

The invention includes a foodstuff, cosmetic, or nutraceuticalcomprising or produced from the ingredient of the current invention.

For example, in the food industry oligosaccharides produced by thecurrent method may be used as sweeteners, bulking agents, added dietaryfibre, or humectants. They may be incorporated into cakes, bread, orother baked goods, or into chocolate or other confectionery such astoffee, fudge, meringue, or caramel; or drinks, for example to providefavourable taste or colour characteristics or to increase dietary fibrecontent. Or they may be incorporated into animal feed, for exampleeither as an isolated ingredient or by utilising the enzymatic reactionmixture directly as feed.

Of particular note is the use as a sweetening agent. As monosaccharidesand disaccharides contribute to dental disease, calorific excess,obesity, and diabetes, and potentially behavioural issues, in certainapplications food manufacturers would prefer not to includemonosaccharides and disaccharides in their products. Theoligosaccharides of the current invention, as their production methodproduces substantially no monosaccharides or disaccharides, may be usedas sweetening agents, allowing foodstuffs to be sweet without exertingthe detrimental effects of monosaccharides and disaccharides.

In the cosmetics industry, monosaccharides and disaccharides maycontribute to spoilage if not removed at some stage of manufacture,while oligosaccharides are useful as ingredients, as they may improvetexture and moisture retention, act as UV-absorbing molecules, maintaina gel or cream structure, and/or serve as bulking agents. Thus, thepresent invention includes a foodstuff, cosmetic, or nutraceuticalcomprising the oligosaccharide-containing ingredient obtainable by themethod of the invention.

The oligosaccharides of the present invention are useful whenincorporated into nutraceutical compositions, as the dietary fibre theyprovide without substantial concomitant provision of dietary sugar hasbeen shown to encourage digestive health, well-regulated gut flora, andother benefits to wellbeing. In this context they may also function asan ingredient in a probiotic drink or other prebiotic or probioticformulation.

EXAMPLES Example 1 Manufacturing Oligosaccharides from Cellulose Usingan LPMO

1. Phosphoric acid-swollen cellulose (PASO) was prepared by making aslurry of 1 g Avicel cellulose (Sigma-Aldrich) with 3 ml H₂O beforeadding 30 ml ice-cold phosphoric acid and incubating at 0° C. for 1 h.The cellulose was then washed numerous times with water until theflowthrough had a neutral pH before use in reactions.

2. Apo-PaLPMO9E (SEQ ID NO:1) was pre-incubated for 0.5-1 h at 5° C. in0.9 stoichiometric Cu(II)(NO₃)₂ immediately before enzyme reactions.

3. 25 μg PASO, 30 μg PaLPMO9E (pre-loaded with copper) and 500 nmolascorbate were incubated in 100 μl 100 mM ammonium acetate pH 6 for 32hours at 50° C. with intermittent shaking.

4. Samples were centrifuged and supernatants were dried in vacuo.

5. Supernatants were reductively labelled with ANTS and analysed by PACE(as per Goubet et al. 2002). FIG. 1 shows the resulting gel.

Example 2 Manufacturing Oligosaccharides from Mixed-Linkage Glucan Usinga Lichenase and Incorporation of Said Oligosaccharides into a Cake

1. 250 g ground porridge oat powder was boiled in 2 I water for 30 min.

2. Once cooled, 2 I ice-cold 96% (v/v) ethanol was added and thesuspension was allowed to sit overnight at 5° C. The suspension wasfilter through miracloth until dry, resuspended in 50% (v/v) ethanol andagain filtered through miracloth.

3. The remaining mass was boiled in 1 I water and incubated for 16 h at30° C. with 2000 U of lichenase from Bacillus subtilis (SEQ ID NO:2,Megazyme).

4. Once cooled, 2 I ice-cold 96% (v/v) ethanol was added and thesuspension was allowed to sit overnight.

5. The supernatant was collected by centrifugation and dried in vacuo,yielding 5.2 g mixed-linkage glucan oligosaccharides. An aliquot wasreductively labelled with ANTS and analysed by PACE. FIG. 2 shows theresulting gel.

6. One medium egg was beaten with 50 g butter and 50 g plain flour.

7. 3 g of the mixture was taken and mixed with 1 g of sugar.

8. 3 g of the mixture was taken and mixed with 1 g of mixed-linkageglucan oligosaccharides.

9. 4 g of the mixture was taken and not mixed further with anything.

10. All three batter mixtures were baked on a baking tray in apre-heated oven at 180° C. for 5 min.

11. After baking, the cakes were cooled, photographed and tasted. FIG. 3shows the photograph.

12. The cake without added sugar or oligosaccharide was unable to holdthe butter inside, which instead leaked out during baking. It has asmooth surface and doughy texture similar to pie pastry, and had asavoury flavour.

The cake containing sugar held butter well and had a more crumbly andspongy texture and surface, characteristic of cakes. It also becamebrown and crisp at the edges. It had a very sweet taste.

Similar to the sugar-containing cake, the cake containing mixed linkageglucan oligosaccharides held butter well and had a characteristicallycake-like texture and surface. It also became brown and crisp at theedges like the sugar-containing cake. It was sweeter than the cakewithout added sugar or oligosaccharides, but not as sweet as the cakecontaining sugar.

Example 3 Manufacturing Oligosaccharides from Xylan Using a GH30Xylanase

1. Spruce wood chips were blended in suspension in a food blender untilthey broke into small particles, and then ball-milled.

2. 100 μl reaction mixtures containing 3.3 mg ball-milled spruce woodchips and 100 mM ammonium acetate pH6 were incubated for 16 h at 30° C.with (or without) 5 μg Ruminiclostridium thermocellum GH30 (sourced fromNZYTech).

3. Reaction products were reductively labelled with ANTS and analysed byPACE. FIG. 4 shows the resulting gel.

Example 4 Manufacturing Oligosaccharides from Xyloglucan Using aXyloglucanase

1. 100 μl reaction mixtures containing 1% (w/v) tamarind xyloglucan and100 mM ammonium acetate pH6 were incubated for 16 h at 30° C. with (orwithout) 0.1 U xyloglucanase (GHS, CAS: 76901-10-5) from Paenibacillussp. (Megazyme).

2. Reaction products were reductively labelled with ANTS and analysed byPACE. FIG. 5 shows the resulting gel.

Prophetic Example 5 Banana Bread Baked Using the Disclosed FoodstuffIngredient

A basic banana bread recipe making 10 servings, consists of one cup (US)(192 g) of sugar (i.e. granulated pure cane sugar for drinks and cereal,such as that provided by Tate and Lyle), 113.5 g of butter, three ripebananas, three eggs, two cups of all-purpose flour, 1 tea spoon ofbaking soda and ½ tea spoon of salt.

An oven is preheated to 190° C. The bananas are mashed in a bowl using afork. In a separate bowl, the flour, baking soda and salt are mixed. Thebutter and sugar are whisked until combined and creamed. The mashedbananas are added and mixed well followed gradually by the whisked eggsuntil well blended. Then, the flour mixture is folded in. The mixture ispoured into a greased baking loaf tin and baked for 45 mins, or until aninserted toothpick comes out clean. The basic bread is cooled on acooling rack. The bread is cut into 10 portions.

Banana bread A is prepared using the same recipe as the basic bananabread, except 30% of the sugar is replaced with the disclosed ingredientof the invention, so 134 g of cane sugar and 58 g of the disclosedingredient of the invention are used.

Banana bread B is prepared using the same recipe as the basic bananabread, except 50% of the sugar is replaced with the disclosed ingredientof the invention, so 96 g of cane sugar and 96 g of the disclosedingredient of the invention are used.

Banana bread C is prepared using the same recipe as the basic bananabread, except 100% of the sugar is replaced with the disclosedingredient of the invention, so 0 g of cane sugar and 192 g of thedisclosed ingredient of the invention are used.

Results

The nutritional values of the banana breads are shown in Table 1. Theseare calculated using USDA National Nutrient Database for StandardReference Legacy Release, April 2018(https://ndb.nal.usda.gov/ndb/search/list?home=true) using the followingrecords: eggs (NDB Id 01123), cane sugar (NDB Id 45167812), butter (NDBId 01145), bananas (NDB Id 09040), all-purpose flour (NDB Id 45054364),baking soda (NDB Id 18372), table salt (NDB Id 02047) and consideringthe whole recipe making 10 servings.

There is an 8% calorie reduction for bread A compared to the basicbread, a 30% reduction of added sugar and a 24% reduction in totalsugar. There is a 12% calorie reduction for bread B compared to thebasic bread, a 50% reduction of added sugar and a 39% reduction in totalsugar. There is a 25% calorie reduction for bread C compared to thebasic bread, a 100% reduction of added sugar and a 79% reduction intotal sugar.

TABLE 1 Nutritional value of one portion of each of the banana breadsdescribed. Basic bread Bread A (one Bread B (one Bread C (one (oneportion) portion) portion) portion) Protein (g) 4.5 4.5 4.5 4.5 Fat (g)10.5 10.5 10.5 10.5 Carbohydrate 45.8 45.8 45.8 45.8 (g) Fiber (g) 1.71.7 1.7 1.7 Sugar (g) 24.4 18.6 14.8 5.2 Calories 291.5 269.9 255.5219.5 (kcal)

REFERENCES

Goubet F, Jackson P, Deery M J, Dupree P. Polysaccharide analysis usingcarbohydrate gel electrophoresis: a method to study plant cell wallpolysaccharides and polysaccharide hydrolases. Anal Biochem. 2002, 53-68

Simmons T J, Uhrin D, Gregson T, Murray L, Sadler I H, Fry S C. Anunexpectedly lichenase-stable hexasaccharide from cereal, horsetail andlichen mixed-linkage β-glucans (MLGs): Implications for MLG subunitdistribution Phytochemistry. 2013, 322-332

Enzyme Sequences LPMO AA9 LPMO from Podospora anserineGenbank ID CAP67740 (SEQ ID NO: 1) 1mkgllsvaal slaysevsah yifqqlstgs tkhgvfqyir qntnynspvt dlssndlrcn 61eggasgantq tvtvragdsf tfhldtpvyh qgpvsvylsk apgsassydg sgtwfkikdw 121gptfpggqwt lagsytaqlp scitdgeyll riqslgihnp ypagtpqfyi scaqikvtgg 181gsvnpsgvai pgafkatdpg ytaniysnfn sytvpgpsvf scgsngggss pvepqpqptt 241tivtstrapv atqpagcava kwgqcggngw tgcttcaags tcntqnayyh qcv. LichenaseGH16 Lichenase from Bacillus subtilis subsp. subtilis str. 168GenBank ID CAA86922.1 (SEQ ID NO: 2) 1mpylkrvlll lvtglfmslf avtatasaqt ggsffdpfng ynsgfwqkad gysngnmfnc 61twrannvsmt slgemrlalt spaynkfdcg enrsvqtygy glyevrmkpa kntgivssff 121tytgptdgtp wdeidieflg kdttkvqfny ytngagnhek ivdlgfdaan ayhtyafdwq 181pnsikwyvdg qlkhtatnqi pttpgkimmn lwngtgvdew lgsyngvnpl yahydwvryt 241kk. Xylanase GH5 Arabinoxylanase from Ruminiclostridium thermocellumGenBank ID ABN53395.1 (SEQ ID NO: 3) 1mgasiktsik irtvafvsii aialsilsfi pnrayaspqr grprinaart tfvgdngqpl 61rgpytstewt aaapydqiar vkelgfnavh lyaecfdpry papgskapgy avneidkive 121rtrelglylv itignganng nhnaqwardf wkfyapryak ethvlyeihn epvawgppys 181sstanppgav dmeidvyrii rtyapetpvl lfsyavfggk ggaaealkdi rafnkavign 241enavwtneav afhgyagwqe ttiaveellk agypcfmtey aggawgsgmg gldveltyel 301erlgvswltf qyipptgvsd dvtkpeyfsa lvensglswt pdygnwpaar gvygngglar 361etatwinnfl tgttrieaed fdwggngvsy ydtdsvnvgg qyrpdegvdi ektsdtgggy 421nvgwisegew leytirvrnp gyynlslrva gisgsrvqvs fgnqdktgvw elpatggfqt 481wttatrqvfl gaglqklrin alsggfnlnw ielspistgt ipdgtykfln rangktlqev 541tgnnsiitad ykgiteqhwk iqhigggqyr issagrgwnw nwwmgfgtvg wwgtgsstcf 601iisptgdgyy rivlvgdgtn lqissgdpsk iegkafhgga nqqwailpvs apafptglsa 661vldssgntan ltwnaapgan synvkrstks ggpyttiatn itstnytdtg vatgtkyyyv 721vsaysngvet lnsaeailqy pkltgtvigt qgswnnignt ihkafdgdln tffdgptang 781cwlgldfgeg vrnvitqikf cprsgyeqrm iggifqgank edfsdavtlf titslpgsgt 841ltsvdvdnpt gfryvrylsp dgsngniael qffgtpagee nddvhlgdin ddgninstdl 901qmlkrhllrs irltekqlln adtnrdgrvd stdlallkry ilrvittl.GH5 Xylanase from Gonapodya prolifera GenBank ID KX518720.1(SEQ ID NO: 4) 1marlsslial vlafvaysap alaargrprl ngktfvadsg vplrgpftst ewtpavpaan 61ianmrnynfn aihlyaetfd pnypaagsqk pgyaatrvdq ivaatkaanm yvvivlanga 121nngkfnlnya kdfwsfyaar yknethviye ihnepvqwgp pyisstqspg aysmnadcyk 181iiravapdtp vllftyasig ggssaagavk daqsfntavf gnanaqwtne aiaihgywga 241qgasdaakal naagfsvvlt efaaatspts pnggqdtvlt gfmeqqgvsw ltflhvpptg 301vsgdvtdpnq ytnrmtaagi gfdrdpglna vgggqaapvp vpapapvpsp vpapvpavpa 361vrtttarpap spspvpapvp apapvpapvp apvpapvpap vpapvpaspa atttrrhrtr 421pprtttapav papppaatpk vcg. GH30 xylanase from Dickeya chrysanthemiGenBank ID AAB53151.1 (SEQ ID NO: 5) 1mngnvslwvr hclhaalfvs atagsfsvya dtvkidanvn yqiiqgfggm sgvgwindlt 61teqintaygs gvgqiglsim rvridpdssk wniqlpsarq ayslgakima tpwsppaymk 121snnslinggr llpanysayt shlldfskym qtngaplyai siqnepdwkp dyescewsgd 181efksylksqg skfgslkviv aeslgfnpal tdpvlkdsda skyvsiiggh lygttpkpyp 241lagnagkqlw mtehyvdskq sannwtsaie vgtelnasmv snysayvwwy irrsygllte 301dgkvskrgyv msqyarfvrp galriqaten pqsnvhltay kntdgkmviv avntndsdqm 361lslnisnanv tkfekystsa slnveyggss qvdssgkatv wlnplsvttf vsk.GH30 xylanase from Bacillus subtilis subsp. subtilis str. 168GenBank ID CAA97612.1 (SEQ ID NO: 6) 1mlprlkktlc vllvcftmls vmlgpgatev laasdvtvnv saekqvlrgf ggmnhpawag 61dltaagreta fgngqnqlgf sllrlhvden rnnwykevet aksavkhgal vfaspwnpps 121dmvetfnrng dtsakrlkyn kyaayaqhln dfvtfmknng vnlyalsvqn epdyahewtw 181wtpqellrfm renagslnar vlapesfqyl knlsdpllnd pqalanmdll gthlygtqvs 241qfpyplfkqk gagkdlwmte vyypnsdtns adrwpealdv sqhlhnamve gdfqayvwwy 301lrrsygpmke dgtlskrgyn mahfskfvrp gyvrldatkn pnanvyvsay kgdnkvvlva 361lnksntgvnq nfvlqngsas nvsrwltsss snlqpgtnlt vsgnhfwahl paqsvttfvv 421nr. GH30 Xylanase from Bacteroides ovatus GenBank ID 5DY64378.1(SEQ ID NO: 7) 1mknltllfcl flanlllgac sggedekkem degkgayalf lkksltystg esqtdvvvew 61aktsweltlg egdlvksvtp tsggsntgek qytkvrvscg anstmkkrtq tlhlfdktne 121ttvdllveqe ppfksvtltv dpsvkyqpvv gfggmynpkl wcgdnllsas qldkmygagg 181lgysllrlml ypnesdwsad veaakaaqan gallfacpwd ctdaladklt vngkemkhlk 241kenyeayanh llryvtfmke kgvnlyalsv qnepdmefty wtpsevvdfv kqygarlret 301gvklmspeac gmqpeytdpl lnnaeafaqt dllaghlyqg ftdlssgyvk nrhdylcgvy 361srlqgktwwm tehlfndgen sddsskwefl kwqyslnhlg kelhmcmegy csaylywylk 421rfyglmgdtd krsptsegel tkngylmahy aqyatettrl kvvtnneevc ataywdektg 481evtlvllnln gasqwlelpl aglkkasave tnetknmevl dtglmesaeg ltyllsansl 541tsvrltf. Xyloglucanase GH5 Xyloglucanase from Bacteroides ovatusGenBank ID ALJ47680.1 (SEQ ID NO: 8) 1mekqsfsdgl fsplgikrvi fmlvllttsf iscsnsdekg gslevaqeyr nlefdargsr 61qtiqidgpae whistseswc ksshtigegk qyvnitvean dtqkertatv tvsasgapdi 121iinvkqslys vpaydeyiap dntgmrdlts mqlsalmkag vnvgntfeav ivgndgslsg 181detcwgnptp nkvlfegika agfdvvripv ayshqfedaa tykiksawmd kveaavkaal 241daglyviini hweggwlnhp vdankealde rleamwkqia lrfrdyddrl lfagtnevnn 301ddangaqpte enyrvqngfn qvfvntvrat ggrnhyrhli vqayntdvak avahftmpld 361ivqnriflec hyydpydfti mpndenfksq wgaafaggdv satgqegdie atlsslnvfi 421nnnvpviige ygptlrdqlt gealenhlks rndyieyvvk tcvknklvpl ywdagytekl 481fdrttgqphn aasiaaimkg ln.GH8 Xylanase from Pseudoalteromonas haloplanktis PDB: 2A8Z_A(SEQ ID NO: 9) 1afnnnpssvg ayssgtyrnl aqemgktniq qkvnstfdnm fgynntqqly ypytengvyk 61ahyikainpd egddirtegq swgmtaavml nkqeefdnlw rfakayqknp dnhpdakkqg 121vyawklklnq ngfvykvdeg papageeyfa fallnasarw gnsgefnyyn daitmlntik 181nklmenqiir fspyidnitd psyhipafyd yfannvtnqa dknywrqvat ksrtllknhf 241tkvsgsphwn lptflsrldg spvigyifng qanpgqwyef dawrvimnvg ldahlmgaqa 301whksavnkal gflsyaktnn skncyeqvys yggaqnrgca gegqkaanav allastnagq 361aneffnefws lsqptgdyry yngslymlam lhvsgnfkfy nntfnGH10 Xylanase from Caldicellulosiruptor owensensis GenBank: ADQ03732.1(SEQ ID NO: 10) 1mseyqdktip slaekykeyf kigaavtvkd legvhgeilv khfnsltpen dmkferihpd 61ehrynfdavd kmkefaiknn mkmrghtfvw hnqtpewvfk dregndvsre llierlrehi 121ktvcdryrdi vyawdvvnea vedktekllr dsnwrriigd dyikiafeia keyagegklf 181yndynnempy klektykllk elidketpid gigiqahwni wdknlidnlk raiemyaslg 241leigiteldm svfefedrrt dllepaeemm elqakvyedv fkvfreykgv itsvtfwgis 301dkhtwkdnfp vigrkdwpllfdvngkpkea ffrivnfGH11 Xylanase from Thermobifida halotolerans GenBank: AEH04392.1(SEQ ID NO: 11) 1mndapahpks rrhgrirlfv grvctalval vtattmlpgv anaavtsnqt gthdgyfysf 61wtdspgtvsm elgpggnyst swsntgnfvv gkgwstggrr tvtysgsfnp sgnayltlyg 121wtrnplveyy ivdnwgtyrp tgtykgtvts dggtydiyet trtnapsieg tatfkqywsv 181rqsrrtggti tagnhfdawa rhgmnlgshd ymimategyq ssgssnitvg gsgggnpggn 241pggnpggggc tatlsagqqw sdrynlgvsv sgssnwtvtm nvpspakiia twnisasypn 301aqtltarpng ngnnwgvtiq hngnwtwptv scsan

1.-23. (canceled)
 24. A sweetening agent comprising: a first β-glucanoligosaccharide with a degree of polymerization (DP) of 3; a β-glucandisaccharide; and glucose, wherein the glucose is present in an amountless than 10% dry w/w.
 25. The sweetening agent of claim 24, wherein theβ-glucan disaccharide is present in an amount less than 40% dry w/w. 26.The sweetening agent of claim 24, further comprising a second β-glucanoligosaccharide with a DP of four.
 27. The sweetening agent of claim 24,further comprising a second β-glucan oligosaccharide with a DP of threeto seven and a third β-glucan oligosaccharide with a DP of three toseven.
 28. The sweetening agent of claim 24, the first β-glucanoligosaccharide comprising at least one 1→4-linkage.
 29. The sweeteningagent of claim 24, the first β-glucan oligosaccharide having only1→4-linkages.
 30. The sweetening agent of claim 24, further comprisingβ-glucan polysaccharides.
 31. The sweetening agent of claim 24, havingless than 5% dry w/w glucose and less than 30% dry w/w β-glucandisaccharides.
 32. The sweetening agent of claim 24, having less than20% dry w/w monosaccharides and less than 60% dry w/w disaccharides. 33.The sweetening agent of claim 24, the sweetening agent substantiallycomposed of saccharides.
 34. The sweetening agent of claim 24, being adry solid.
 35. The sweetening agent of claim 24, further comprisingascorbic acid, gallic acid, cysteine, NADH, NADPH, pyrogallol,dithiothreitol, cyanoborohydrides, borohydrides, photosyntheticpigments, lignin, lignol, or a combination thereof
 36. The sweeteningagent of claim 24, further comprising lactone, 4-ketoaldose, aldonicacid, geminal diol, or a combination thereof.
 37. The sweetening agentof claim 24, further comprising an oligosaccharide that is not aβ-glucan oligosaccharide.
 38. The sweetening agent of claim 37, theoligosaccharide that is not a β-glucan oligosaccharide having at leastone side chain.
 39. The sweetening agent of claim 24, wherein when thesweetening agent is incorporated into a foodstuff, the foodstuff has acomparable texture to a control foodstuff, and wherein the controlfoodstuff is comparable to the foodstuff except that the controlfoodstuff comprises sucrose as a sweetener component.
 40. The sweeteningagent of claim 24, wherein when the sweetening agent is incorporatedinto a foodstuff, the foodstuff has at least 5% fewer calories than acontrol foodstuff, and wherein the control foodstuff is comparable tothe foodstuff except that the control foodstuff comprises sucrose as asweetener component.
 41. The sweetening agent of claim 24, wherein whenthe sweetening agent is incorporated into a foodstuff, the foodstuff hasa greater fiber content than a control foodstuff, and wherein thecontrol foodstuff is comparable to the foodstuff except that the controlfoodstuff comprises sucrose as a sweetener component.
 42. The sweeteningagent of claim 24, wherein the sweetening agent is incorporated into abaked good, a chocolate, a confectionery, or a drink.
 43. The sweeteningagent of claim 24, wherein when the sweetening agent is incorporatedinto a foodstuff the first β-glucan oligosaccharide is oxidized orreduced.